Combination bacteriolytic therapy for the treatment of tumors

ABSTRACT

Current chemotherapeutic approaches for cancer are in part limited by the inability of drugs to destroy neoplastic cells within poorly vascularized compartments of tumors. We have here systematically assessed anaerobic bacteria for their capacity to grow expansively within avascutar compartments of transplanted tumors. Among 26 different strains tested, one ( Clostridium novyi ) appeared particularly promising. We created a strain of  C. novyi  devoid of its lethal toxin ( C. novyi -NT) and showed that intravenously injected  C. novyi -NT spores germinated within the avascular regions of tumors in mice and destroyed surrounding viable tumor cells. When  C. novyi -NT spores were administered together with conventional chemotherapeutic drugs, extensive hemorrhagic necrosis of tumors often developed within 24 hours, resulting in significant and prolonged anti-tumor effects. This strategy, called combination bacteriolytic therapy (COBALT), has the potential to add a valuablle dimension to the treatment of cancer.

This invention was made using U.S. government support from NIH grants CA43460 and CA 62924. The U.S. government therefore retains certain rightsin the invention.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the field of oncology. In particular itrelates to combined biological and chemical treatments of tumors.

BACKGROUND OF THE INVENTION

Despite enormous progress in understanding the pathophysiology ofneoplasia, advanced forms of cancer remain recalcitrant to treatment.Though the basis for this failure is complex, one reason is that mosttumors contain large, poorly vascularized areas that limit the efficacyof radiation and chemotherapeutic drugs (Jain, 1994)(Jain, 2001). Thepoorly vascularized regions are less sensitive to ionizing radiationbecause its cell-killing effects are dependent on oxygen; they are lesssensitive to chemotherapeutic drugs because drug delivery to theseregions is obviously suboptimal. As a cancer therapeutic agent must notleave significant clusters of viable cells within every lesion toachieve a clinically meaningful effect, the poorly vascularized regionsof tumors represent a major obstacle to effective treatment.

One of the most important recent developments in tumor biology is therecognition that neoangiogenesis is essential for the growth of tumorsto clinically meaningful sizes. What is less well-recognized is thatthis neoangiogenesis often does not keep pace with the growth of theneoplastic cells, resulting in large necrotic areas composed of dead ordying cells. For example, we found that each of 20 randomly selectedliver metastases >1 cm³ in size contained relatively large regions ofnecrosis/apoptosis, in general constituting 25% to 75% of the tumor mass(FIG. 1). Cells adjacent to these necrotic areas are poorly vascularizedand likely to be difficult to treat with conventional agents.

It has been recognized for half a century that anaerobic bacteria canselectively proliferate in the hypoxic regions of tumors (Parker,1947)(Malmgren, 1955)(Mose, 1963)(Gericke, 1963)(Thiele, 1963)(Carey,1967)(Kohwi, 1978)(Brown, 1998)(Fox, 1996)(Lemmon, 1997)(Sznol,2000)(Low, 1999)(Clairmont, 2000)(Yazawa, 2000)(Yazawa, 2001)(Kimura,1980). Clever strategies for potentially exploiting such bacteria fordiagnostic and therapeutic purposes have been devised, though relativelylittle work in this area has recently taken place.

SUMMARY OF THE INVENTION

One embodiment of the invention provides a method for treating tumors ina mammal. Spores of an anaerobic bacterium are administered to themammal. A toxin gene of a wild type form of the anaerobic bacteriwn isdeleted in the spores of the anaerobic bacterium, rendering the sporesof the anaerobic bacterium less toxic to the mammal. An anti-tumor agentis also administered to the mammal. The tumor regresses or its growth isslowed or arrested as a result of these administrations.

Another embodiment of the invention provides a kit for treating tumors.The components of the kit are in a divided or undivided container. Thecomponents include spores of an anaerobic bacterium which istoxin-defective and an agent which collapses tumor vasculature.

Also provided by another embodiment of the present invention is anisolated and bacteriologically pure Clostridium novyi bacterium which istoxin-defective.

Still another embodiment of the invention provides an isolated andbacteriologically pure Clostridium sordelii bacterium which istoxin-defective.

These and other embodiments of the invention which will be apparent tothose of skill in the art upon reading the specification provide the artwith an exciting modality for treating patients with tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Typical human colorectal metastases. Extensive areas ofnecrosis, indicated with arrows, are intermixed with areas of viabletumor cells. Similar large areas of necrosis were observed in each ofthe metastatic lesions from 20 different patients chosen at random fromthe pathologic archives.

FIGS. 2A -2D. Distribution of anaerobic bacteria within tumors. Micebearing subcutaneous B16 tumors were intravenously injected through thetail vein with 5×10⁷ live B. longum bacteria or wild-type C. novyispores. Mice with B. longum were given intraperitoneal injection withlactulose daily for five days to increase bacterial growth (Yazawa, 2000) and then sacrificed for analysis of tumor colonization. Mice with C.novyi were sacrificed the day after injection for analysis. Gram-stainsrevealed that a large number of B. longum bacteria was concentratedwithin a few colonies while C. novyi was dispersed throughout the poorlyvascularized portions of the tumors. (FIG. 2A, FIG. 2B) High and lowpower view of representative B. longum experiment, showing bacteria(stained deep blue) clustered within a colony. (FIG. 2C) C. novyiexperiment, showing dispersion of bacteria throughout the necroticregion of the tumor. (FIG. 2D) High power view, showing invasion of C.novyi bacteria into surrounding viable tumor cells (stained purple) onthe left.

FIG. 3. Elimination of the lethal toxin gene from C. novyi. Followingheat shock, PCR was performed on DNA from colonies to identify thosewhich had lost the lethal toxin gene on the phage episome. Agarose gelelectrophoresis of the PCR products with two independent primer sets(ToxA and ToxB) shows a C. novyi clone (C. novyi-N7) that had lost thegene and a clone (C. novyi) which retained them. Controls were providedby primer sets (PlcA and PlcB) specific for the C. novyi phospholipase Cgene demonstrating the integrity of the DNA templates in all reactions.

FIG. 4A-4D. Distribution of C. novyi-NT bacteria after intravenousinjection of spores.

(FIG. 4A) H & E stain of a typical HCT116 tumor xenograft from a mousenot injected with bacteria, showing some necrosis. (FIG. 4B) H & E stainof a tumor 24 hours following intravenous injection of 5×10⁷ C. novyi-NTspores. (FIG. 4C) Gram stain, revealing bacteria distributed throughoutthe necrotic region. (FIG. 4D) High power view, showing a lawn ofbacteria at the interface between the viable and necrotic regions of thetumor at the top and bottom of the picture, respectively.

FIGS. 5A -5B. Hemorrhagic necrosis following COBALT. (FIG. 5A) HCT116tumor-bearing mouse 24 hours after iv injection with 5×10⁷ C. novyi-NTspores. Slight swelling associated with edema is seen at the tumor site.D10 (0.3 mg/kg) was then given intravenously (time=0), and followed 24hours later with MMC (4 mg/kg). A black spot indicating hemorrhagicnecrosis is evident near center of tumor at 0.3 days. The area ofhemorrhagic necrosis gradually expanded over the next day. Swelling atthe tumor site then resolved and the necrotic tumor mass and skinoverlying it shrunk and gradually dissolved (days 6 to 30). (FIG. 5B)Typical mice five weeks after treatment with a single dose of D10 plusMMC (top) or with a single dose of COBALT (bottom). Of the eight micetreated with COBALT in this experiment, four were apparently cured oftheir tumor, as no recurrence was observed after three months ofobservation.

FIGS. 6A -6C. Quantitation of the Effects of COBALT. (FIG. 6A) HCT116colorectal cancer cells were grown as xenografts in nude mice. When thetumors were ˜700 mm³ in size, the animals were injected intravenouslywith 5×10⁷ C. novyi-NT spores (time 0), followed by iv injection withD10 (0.3 mg/kg) at 24 hours and ip injection with MMC (4 mg/kg) at 48hours. Control groups were given no treatment or treated with D10 plusMMC without spores. Each group consisted of six to ten mice. Animalswere euthanized when their tumors exceeded 15% of their body weight. Inthe experiment shown, seven of eight mice treated with a single dose ofCOBALT developed a striking hemorrhagic necrosis of their tumors within24 hours after administration of D10. Four of these seven mice werecured, while three of the mice died three days after treatment, perhapsfrom tumor lysis syndrome (see Discussion). One mouse developed lessextensive necrosis and its tumor eventually regrew. Only mice thatsurvived treatment were used to obtain the data plotted in the graph.(FIG. 6B) Mice were treated as in (FIG. 6A), except that MMC was notused and treatments were given once every two weeks. (FIG. 6C) B16melanoma cells were grown as subcutaneous syngeneic tumors in C57BL/6mice. When the tumors were approximately 700 mm³ in size, the animalswere injected intravenously with 5×10⁷ C. novyi-NT spores (time 0),followed by ip injection with CTX (100 mg/kg) at 6 hours and ivinjection with D10 (0.3 mg/kg) at 24 hours. Other groups were given notreament, CTX plus D10, or spores plus D10. Each group consisted of atleast ten mice and the treatments were repeated at weekly intervals.Four mice died after the first dose of COBALT and only those mice thatsurvived treatment were used to obtain the data plotted in the graph.Animals were euthanized when their tumors exceeded 15% of body weight.

DETAILED DESCRIPTION

It is a discovery of the present inventors that combinationbacteriolytic therapy (COBALT) can result in rapid and dramaticregressions of experimental tumors in mice. Even relatively large tumorscould be treated successfully with COBALT, even though such tumors donot generally respond well to chemotherapeutic agents.

The bacteria useful in the practice of the present invention areanaerobic, spore formers. Suitable genera include Bifidobacteria,Lactobacilli, and Clostridia. A number of species of these bacteria havebeen tested for their ability to grow in tumors in a robust anddispersed manner. Clostridium novyi and Clostridium sordelii were foundto be the best of the strains we tested for these properties. Otherstrains and species having suitable characteristics can be used as well.

Decreasing the natural production of toxins is desirable in usingbacteria therapeutically. While strains need not be totallynon-toxigenic, it is desirable that at least one of the toxin genes bymutated, deleted, or otherwise inactivated to render the bacteria lessharmfull to the host. If a toxin gene is episomal or on a phage, thencuring of the episome or phage can be used to delete the toxin gene.Techniques are well known in the art for mutagenesis and screening ofmutants.

Isolated and bacteriologically pure vegetative bacteria or spores,according to the invention are those which are not contaminated withother bacteria or spores. Microbiological techniques for obtaining suchpure cultures are will known in the art. Typically single colonies arepicked and spread upon an agar nutrient medium, separating colonies sothat new colonies arise that are the progeny of single cells. Thisprocess is typically repeated to ensure pure cultures. Alternatively,liquid cultnies can be serially diluted and plated for single colonyformation. Serial repitition is desirable to enure colony formation fromsingle cells. See, e.g., J. H. Miller, Experiments in MolecularGenetics, Cold Spring Harbor Laboratory, NY, 1972.

Spores can be administered to a tumor-bearing mammal by any means whichwill afford access to the tumor. Spores can be injected intravenously,intradermally, subcutaneously, intramuscularly, intraperitoneally,intratrumorally, intrathecally, surgically,. etc. Preferred techniquesare intravenous and intratumoral injections. Tumor bearing mammals canbe humans, pets, such as dogs and cats, agricultural animals such ascows, sheep, goats and pigs, and laboratory animals, such as rats,hamsters, monkeys, mice, and rabbits. The tumors to be treated arepreferably large enough to have outgrown their blood supply and containnecrotic regions. This factor should not be limiting for most humantumor situations, as the great majority of clinically apparent humantumors have large necrotic regions within them (FIG. 1). However,micrometastatic disease might not be susceptible to COBALT.

Combination treatment involves administering anaerobic spores as well asa second anti-tumor agent. Together these agents synergize to produce agreater decerease in the growth of the tumor. Second anti-tumor agentswhich can be used include any which are known in the art. Suchanti-tumor agents include but are not limited to DNA damaging agents,agents which collapse tumor vasculature, radiation, and anti-tumorantigen antibodies. These anti-tumor agents are administered accordingto the conventional means used in the art of medical and radiationoncology. The agents can be administered in any order or simultaneously.It may be desirable, however, to administer the spores prior toadministering the second anti-tumor agent. If agents are to beadministered serially, they are preferably administered within a span ofa month, more preferably within a span of a fortnight, and even morepreferably within a span of a week. Optimization of the time span iswell within the skill of the art. Moreover, multiple anti-tumor agentscan be administered in conjunction with the spores. Thus it may bedesirable in order to achieve even greater reduction in tumor growththat a plurality of anti-tumor agents be used. Anti-tumor agents fromdifferent categories or mechanisms may achieve superior results. Thus apreferred combination includes spores, a tumor vasculature collapsingagent and a DNA damaging agent.

Suitable anti-tumor agents which function to collapse tumor vessels arevinblastine, vincristine, colchicine, combrestatin A-4, dolastatin-10,and 5,6 dimethylxanthenone-4-acetic acid. Others as are known ordiscovered with the same function can be used. Suitable DNA damagingchemotherapeutic drugs which can be used include but are not limited tomitomycin C and cytoxan.

In order to mitigate the side-effects of the anti-tumor therapy variousadditional drugs or therapies can be utilized. These includeallopurinol, hydration, uate oxidase, steroids such as prednisone, andhematopoietic factors such as granulocyte colony stimulating factor(G-CSF).

Kits comprising the useful components for practicing the anti-tumormethods of the present invention can be packaged in a divided orundivided container, such as a carton, bottle, ampule, tube, etc. Thespores and anti-tumor agents can be packaged in dried, lyophilized, orliquid form. Additional components provided can include vehicles forreconsititution of dried components. Preferably all such vehicles aresterile and apyrogenic so that they are suitable for injection into amammal without causing adverse reactions. The anti-tumor agents otherthan the spores are also preferably sterile. The spores are preferablymicrobiologically pure, i.e., containing no other bacteria other thanthe desired spore-forming anaerobe.

Treatment of mice with large tumors was sometimes toxic. Approximately20% of mice with 350 mm³ tumors and 50% of mice with 700 mm³ tumors diedwithin 24-72 hours of administration of spores plus D10. No deaths wereobserved after treatment with C. novyi-NT spores alone or with D10alone. Though the basis for this toxicity is not yet known, it couldhave been due to efflux of toxic bacterial products from the tumors ordue to “tumor lysis syndrome.” It has previously been noted that therapid lysis of very large tumor burdens is associated with systemictoxicity in humans treated with chemotherapy, perhaps due to the suddenefflux of tumor cell metabolites into the circulation (Altman, 2001).Though tumor lysis syndrome can be controlled in humans, it is difficultto control in mice. Any therapy which dramatically shrinks tumors may besubject to this side effect. Treatments for tumor lysis syndrome whichmay be used in humans include allopurinol, urate osidase, and volumerepletion (hydration). Treatments to mitigate side-effects of anti-tumoragents such as bone marrow toxicity and neutropenia may also bedesirable. Such treatment are will known in the art and can be employedhere in the known manner.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples which are provided herein for purposes of illustrationonly, and are not intended to limit the scope of the invention.

EXAMPLE 1

In the work described below we attempted to exploit the fact thatnecrotic regions exist only within tumors and in no normal tissues. Wewished to develop a toxic agent that could be specifically delivered tothese areas and, in theory, could kill surrounding viable tumor cells.We chose to investigate anaerobic bacteria for this purpose. We hopedthat a systematic screen for appropriate anaerobic bacteria that couldkill tumor cells adjacent to the poorly vascularized regions, ratherthan just localize to such regions, would rejuvenate interest in thisapproach. Furthermore, we hoped that chemotherapeutic agents that killedthe well-vascularized regions of tumors, when administered inconjunction with appropriate bacteria, would result in the destructionof a major proportion of neoplastic cells within the tumors.

We used the following materials and methods in our studies. Bacterialstrains and growth. The bacterial strains tested in this study werepurchased from the American Type Culture Collection and listed inTable 1. They were grown anaerobically in liquid cultures at 37° C. inReinforced Clostridial Medium or Lactobacilli MRS broth (Difco, Detroit,Mich.).

Drugs. D10 (D10) was kindly provided by Dr. George R. Pettit (CancerResearch Institute, Arizona State University, AZ), Dr. Gregory P.Kalemkerian (Department of Internal Medicine, Wayne State University,MI), and Dr. Robert J. Schultz (Drug Synthesis and Chemistry Branch,NCI, Bethesda, Md.). Combretastatin A-4 was kindly provided by Dr.Robert J. Schultz. Cytoxan (CTX), mitomycin C (MMC), vincristine,colchicine, and vinblastine are commercially available chemotherapeuticagents (Sigma, St. Louis, Mo.).

Cell lines and animals. Female athymic nude and C57BL/6 mice 6 to 8weeks of age were purchased from Harlan. HCT116 colon cancer cells andB16 melanoma cells were grown as monolayers in McCoy SA medium (LifeTechnologies, Rockville, Md.) supplemented with 5% fetal bovine serumand 1% penicillin/streptomycin (Cat. No. 15140-122).

Sporulation and generation of nontoxigenic C. novyi strain. Spores ofboth wild-type and nontoxigenic C. novyi strains were generated bygrowing the organisms anaerobically at 37° C., pH 7.4 in a mediumcontaining 5 g Na₂HPO₄, 30 g peptone, 0.5 L-cysteine, 10 g maltose and5% w/v dried cooked meat particles (Difco, Detroit, Mich.) per 1 liter.After one week in this medium, spores settled in the cooked meatparticle layer (Bagadi, 1973). Spores were further purified fromcontaminating vegetative forms on a discontinuous Percoll gradient. Toremove the lethal toxin gene from the wild type C. novyi strain, C.novyi spores were heated at 70° C. for 15 minutes to inactivate thephage carrying the toxin (Eklund, 1974 )(Eklund, 1976). The spores werethen plated on RCM agar and incubated anaerobically at 37° C. for 48hours. Isolated colonies were cultured in liquid RCM for another 24 to48 hours and then tested for the presence of the lethal toxin gene byPCR. In general, 1% of the bacterial colonies were found to lose thephage carrying this gene.

In vivo studies. Six to eight week old female Balb/c athymic nude orC57BL/6 mice were implanted with subcutaneous tumors through theinjection of 2.5×10⁶ HCT116 or B16 cells, respectively. After 8 to 12days of tumor establishment, treatment was initiated with spores ordrugs. Screening of bacterial strains for their ability to populatetumor grafts was done by either intratumoral injection (100 ul volume,1×10⁷ bacteria) or intravenous injection (500 ul volume, 5×10⁷ bacteriaor spores) of the tail vein. C. novyi-NT spores and D10 were diluted tothe appropriate concentration in 1×Dulbecco's phosphate-buffered salinepH 7.4 (PBS) (Life Technologies, Rockville, Md.) and then administeredby intravenous injection in a volume of 500 ul. CIX and MMC were dilutedin PBS and then given by intraperitoneal injection in a volume of 500ul. Tumor growth was assessed by measuring the size of the major andminor axes of subcutaneous tumors every two and every four days for B16and HCT116 tumors, respectively, using calipers. Tumor volume was thencalculated using the equation length×width²×0.5.

EXAMPLE 2

Choice Of Bacterial Species. From previous studies it was clear thatspecies of anaerobic bacteria could grow within the hypoxic regions oftumors. An example is provided by B. longum, which, when injectedintravenously into mice with subcutaneous tumors, grew specifically androbustly within the tumors but not within normal tissues (Yazawa,2000)(Yazawa, 2001). Gram stains of sections of the tumors, however,revealed that most bacteria were tightly clustered within coloniesrather than distributed throughout the necrotic regions (FIG. 2A, FIG.2B). As we considered dispersion of the bacteria essential to achievethe desired effects, numerous anaerobic species of three differentgenera were tested in an effort to find one(s) exhibiting this phenotype(Table 1). For this purpose, Bifidobacterium and Lactobacillus strainswere injected intravenously, while Clostodium strains, which aregenerally highly toxic when injected intravenously, were injecteddirectly into tumors. Among the 26 strains listed in Table 1, only two(C. novyi and C. sordellii) exhibited extensive spreading throughout thepoorly vascularized portions of the tumors (not shown). Though thisspread was undoubtedly facilitated by the motile nature of these twospecies, other motile anaerobic bacteria, including other Clostridiumstrains, did not exhibit this property when tested under identicalconditions. TABLE 1 Bacterial strains tested Bifidobacteria B.adolescentis ATCC 15703 B. animalis ATCC 25527 B. bifidum ATCC 11863,15696 B. boum ATCC 27917 B. breve ATCC 15700 B. coryneforme ATCC 25911B. dentium ATCC 15423, 27534 B. indicum ATCC 25912 B. infantis ATCC15702, 25962 B. longum ATCC 15707 B. magnum ATCC 27540 B. pseudolongumATCC 25526 Lactobacilli L. bifidus ATCC 11146 L. delbruecki ATCC 21815Clostridia C. absonum ATCC 27555 C. acetobulylicum ATCC 824 C.bifermentans ATCC 17836 C. difficile ATCC 700057 C. histolyticum ATCC19401 C. novyi ATCC 19402 C. perfringens ATCC 3624, 13124 C. sordelliiATCC 9714

EXAMPLE 3

Infiltration of the Tumor Mass Following Intravenous Injection of C.novyi spores. In order for an experimental therapy to represent apotentially viable tool for the treatment of disseminated cancers, itmust have the capacity to be delivered systemically rather than throughlocal, intratumoral injection. Though live bacteria are often toxic wheninjected intravenously, it has been shown that bacterial spores arenon-toxic to normal animals. Accordingly, we found that large numbers(up to 10⁸ in a volume of 500 ul) of C. novyi and C. sordellii sporescould be injected intravenously into normal mice without causing anynoticeable side effects. When intravenously injected into mice withsubcutaneous B16 tumors, however, the C. novyi bacteria floridlygerminated within the tumors within 16 hours (FIG. 2C). In contrast, nogerminated bacteria were observed in the liver, spleen, kidney, lung, orbrain of these mice (not shown). Similar results were observed after ivinjection of C. sordellii spores (not shown).

EXAMPLE 4

Genetic modification of C. novyi. Though C. novyi and C. sordelliispores both had the capacity to grow within tumors and kill somesurrounding tumor cells, there was at least one small problemencountered with this experimental treatment: 16 to 18 hours followingthe initiation of treatment, all the mice died. We suspected that thecause of death was the release of potent lethal toxins from the bacteriagerminating within the tumors. Indeed, other anaerobic bacterial sporeshave proved highly toxic to animals and humans following germinationwithin the anaerobic environments present in tumors or wounds, and theresultant mortality shown to be due to specific secreted toxins (Boyd,1972)(Boyd, 1972)(Bette, 1991)(Rood, 1991)(Bryant, 2000).

To mitigate systemic toxicity, we attempted to eliminate the lethaltoxin gene from C. novyi. We chose C. novyi rather than C. sordellii forthis purpose because the latter has two homologous toxin genes(Martinez, 1992) rather than one and because the single C. novyi toxingene is located within a phage episome (Eklund, 1974)(Eklund,1976)(Hofmann, 1995). Bacteria were heat treated to induce loss of thephage and inoculated onto agar plates. Of 400 bacterial coloniesscreened, three were observed to have lost the toxin gene when assessedby PCR using toxin-gene specific primers (examples in FIG. 3).Phospholipase C, a C. novyi gene contained within the bacterial ratherthan the phage genome (Tsutsui, 1995), served as control for this PCRexperiment. One clone, named C. novyi-NT, that had lost the toxin gene,was selected for further analysis.

EXAMPLE 5

Destruction of Tumor Cells Following Injection of C. novyi-NT spores. C.novyi-NT spores devoid of the lethal toxin were injected intravenouslyinto mice with tumors. These spores retained their capacity to germinatewithin tumors and resulted in greatly expanded areas of necrosis (FIG.4A vs. FIG. 4B). However, these spores, unlike those of their parents,were non-toxic when injected alone, with no ill effects generallyobserved after injection of up to 10⁸ spores into mice with tumors. Incontrast, all mice died after injection of 5×10⁷ parental C. novyispores into mice with tumors. Growing bacteria could be observedthroughout the much-enlarged necrotic regions of tumors after injectionof spores (FIG. 4C, FIG. 4D). The enlargement of the necrotic regionswas apparently due to the destruction of viable tumor cells adjacent tothe original necrotic regions by the bacteria. Indeed, a bacterial“film” (McManus, 1982) was routinely observed at the interface betweenthe necrotic area and the remaining viable rim of the tumor, as if thebacteria were destroying the viable tumor cells and using itsdegradation products as nutrients (FIG. 4D). This tumor infiltrationeffect was similar to that observed with wild-type C. novyi bacteria(FIG. 2D).

EXAMPLE 6

Combination Therapy. We hoped to combine a bacterial agent with moreconventional chemotherapeutic agents in an effort to attack the tumorsfrom both the inside and outside, respectively. Following preliminaryinvestigations with several such agents, we concentrated on two classes:(i) DNA damaging agents, such as MMC and CTX, which selectively killtumor cells, and (ii) agents that appear to partially collapse tumorvasculature, such as flavone acetic acid derivatives and microtubulebinding agents (Chaplin, 1996)(Sweeney, 2001). The latter class ofagents has been shown to be able to interfere with proper circulationthrough the tumors and thereby trap large molecules, such as antibodiesor bacteria, that have gained access to the tumor tissue (Theys,2001)(Pedley, 1999)(Pedley, 2001). Among flavone acetic acid and themicrotubule-binding agents tested (including vinblastine, vincristine,colchicine, combretastatin A-4, and D10), D10 appeared to have the mostpronounced effects and was chosen for further experimentation.

Xenografts of the colorectal cancer cell line HCT116 were used to testthe effects of this combination therapy in nude mice, as the tumorscould easily be visualized under the hairless skin. As shown in FIG. 5,sequential treatment with C. novyi-NT spores, D10 and MMC resulted indramatic effects on large subcutaneous tumors (starting tumor volume˜700 mm³), easily observable through the skin. Twenty four hoursfollowing the injection of C. novyi-NT spores, the tumor mass swelledand became edematous (FIG. 5A). Six hours after receiving D10, a blackspot developed near the center of the tumor, representing an area ofhemorrhagic necrosis. This spot expanded in size and within 24 hoursoften completely enveloped the tumor (FIG. 5A, 1 day time point). H & Estaining of sections of these tumors revealed extensive destruction ofthe tumors, often accompanied by infiltration of inflammatory cells.These necrotic masses then shrank over a period of two to four weeks(FIG. 5A, 14-30 day time points). In many mice, these necrotic masseseventually dissolved and disappeared, leaving the animals tumor-free(FIG. 5B). Similar, though less dramatic results, were observedfollowing the sequential treatment with C. novyi-NT and D10 (withoutMMC), but never with D10 and MMC in the absence of C. novyi-NT andrarely with C. novyi-NT alone.

The anti-neoplastic effects of this combination bacteriolytic therapy(COBALT) were further quantified in the experiments shown in FIG. 6.Animals with relatively large subcutaneous HCT116 tumors (starting tumorvolume ˜700 mm³) were treated with drugs alone (D10 plus MMC) or C.novyi-NT spores plus the drugs. As can be seen in FIG. 6A, the drugsalone slowed the growth of the tumors, though the tumors continued togrow and the animals had to be sacrificed at ten to fourteen days, whentumor weights exceeded 10% of body weight. The addition of C. novyi-NTspores dramatically enhanced the effects of treatment, with tumorsactually shrinking rather than simply slowing. In the experiment shownin FIG. 6A, four of eight animals had complete tumor regressions afteronly one administration of COBALT. Significant tumor shrinkage was alsoseen when mice were given sequential treatment with C. novyi-NT sporesplus D10 (FIG. 6B). However, there was no long-term tumor-free survivaland the treatment had to be repeated once every two weeks unless thefull combination, with MMC, was included. Systemic treatment with C.novyi-NT spores alone slowed tumor growth while D10 alone had no effect,clearly illustrating the value of the combination (FIG. 6B).

To determine whether COBALT would affect other tumor types, we treatedC57BL/6 mice with large syngeneic B16 tumors. In this case, CTX wassubstituted for MMC, as B16 tumor cells were more sensitive to CTX thanto MMC. As shown in FIG. 6C, the drugs alone had some anti-tumoreffects, as expected, though the tumor continued to grow in size and theanimals had to be sacrificed within a week after beginning therapy. C.novyi-NT spores considerably enhanced these effects, and the tumorsremained small over the four week course of this experiment. As withHCT116 human tumors, we found that D10 plus C. novyi-NT spores hadsignificant anti-neoplastic effects on B16 tumors, but that the additionof a tumor cytotoxic agent (CTX) further enhanced the efficacy of COBALT(FIG. 6C). In the B16 tumor model, maintenance COBALT (once weekly) wasrequired to keep tie tumors from regrowing while with HCT116 cells, asingle treatment cured ˜half the mice.

The results recorded above show that COBALT can result in rapid anddramatic regressions of experimental tumors in mice. Even relativelylarge tumors could be treated successfully with COBALT, though tumors ofthe size used in our experiments don't generally respond well tochemotherapeutic agents (FIGS. 6A-6C).

It is also clear that many questions remain. For example, we don'tunderstand the basis for the potent tumor cell killing in the vicinityof the germinating bacteria. We found that many other bacterial strainscould germinate within the necrotic regions of tumors but did notexhibit this potent cytotoxic activity. This killing is clearly not dueto the lethal toxin gene of C. novyi, as this gene was deleted in the C.novyi-NT strain used in COBALT. It will be interesting in the future todetermine which of the C. novyi-NT genes are responsible for these tumorcytolytic effects.

Another point of interest was that an agent acting on the vasculature(D110) was synergistic with the C. novyi-NT spores in causingsignificant tumor shrinkage. Presumably, the vascular collapse furtherlowered the oxygen tension near the trapped bacteria and therebyincreased the potential for bacterial growth. D10 was given after thebacterial spores rather than before because we believed that partialvascular collapse prior to spore administration might have a deleteriouseffect on spore delivery. This belief was based on the fact that othervascular collapsing agents, such as DMXAA and combretastatin A-4, havebeen shown to exert their effects in combination with radioactivelylabeled antibodies only when administered after, and not before theantibodies (Theys, 2001)(Pedley, 1999)(Pedley, 2001).

References

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1. An isolated and bacteriologically pure Clostridium novyi bacteriumwhich is toxin-defective.
 2. The bacterium of claim 1 which is cured ofa bacteriophage which encodes the toxin for which the bacterium isdefective.
 3. The bacterium of claim 1 wherein a toxin gene of wild typeClostridium novyi is deleted rendering the bacterium less toxic to themammal.
 4. The bacterium of claim 1 which is in spore form.
 5. Thebacterium of claim 1 which is in vegetative form.
 6. A method fortreating tumors in a mammal, comprising: administering to the mammalspores of the bacterium of claim 1, whereby the tumor regresses or itsgrowth is slowed or arrested.
 7. An isolated and bacteriologically pureClostridium novyi bacterial spore which is cured of a bacteriophagewhich encodes a toxin.
 8. An isolated and bacteriologically pureClostridium sordelii bacterium which is toxin-defective.
 9. Thebacterium of claim 1 which is in spore form.
 10. The bacterium of claim1 which is in vegetative form.
 11. A method for treating tumors in amammal, comprising: administering to the mammal spores of the bacteriumof claim 8, whereby the tumor regresses or its growth is slowed orarrested.
 12. A method for treating tumors in a mammal comprising:administering to the mammal spores of an anaerobic bacterium in which atoxin gene of a wild type form of the anaerobic bacterium is deleted,rendering the spores of the anaerobic bacterium less toxic to themammal; and administering to the mammal an anti-tumor agent; whereby thetumor regresses or its growth is slowed or arrested.
 13. The method ofclaim 12 wherein the anaerobic bacterium is Clostridium novyi.
 14. Themethod of claim 12 wherein the anaerobic bacterium is Clostridiumsordellii.
 15. The method of claim 12 wherein the spores areadministered intravenously.
 16. The method of claim 12 wherein thespores are administered intratumorally.
 17. The method of claim 12wherein the anti-tumor agent is radiation.
 18. The method of claim 12wherein the anti-tumor agent is an antibody.
 19. The method of claim 12wherein the anti-tumor agent collapses tumor vasculature.
 20. The methodof claim 19 wherein the anaerobic bacterium is Clostridium novyi. 21.The method of claim 19 wherein the anaerobic bacterium is Clostridiumsordellii.
 22. The method of claim 19 wherein the spores areadministered intravenously.
 23. The method of claim 19 wherein thespores are administered intratumorally.
 24. The method of claim 19wherein the anti-tumor agent is vinblastine.
 25. The method of claim 19wherein the anti-tumor agent is vincristine.
 26. The method of claim 19wherein the anti-tumor agent is colchicine.
 27. The method of claim 19wherein the anti-tumor agent is combretastatin A-4.
 28. The method ofclaim 19 wherein the anti-tumor agent is dolastatin-10.
 29. The methodof claim 19 wherein the anti-tumor agent is 5,6dimethylxanthenone-4-acetic acid.
 30. The method of claim 19 furthercomprising: administering a DNA damaging chemotherapeutic drug.
 31. Themethod of claim 30 wherein the DNA damaging chemotherapeutic drug ismitomycin C.
 32. The method of claim 30 wherein the DNA damagingchemotherapeutic drug is cytoxan.
 33. The method of claim 12 wherein thespores and anti-tumor agent are administered serially.
 34. The method ofclaim 30 wherein the spores and agent and DNA damaging chemotherapeuticdrug are administered serially.
 35. A method for treating tumors in amammal comprising: intravenously administering to the mammal spores of aClostridium novyi bacterium which is toxin-defective; administeringdolastatin-10 to the mammal; whereby the tumor regresses or its growthis slowed or arrested.
 36. A method for treating tumors in a mammalcomprising: intravenously administering to the mammal spores of aClostridium novyi bacterium which is toxin-defective; administeringdolastatin-10 to the mammal; and administering cytoxan to the mammal;whereby the tumor regresses or its growth is slowed or arrested.
 37. Themethod of claim 12 further comprising: administering allopurinol to themammal.
 38. The method of claim 12 further comprising: hydrating themammal.
 39. The method of claim 12 further comprising: administeringurate oxidase to the mammal.
 40. The method of claim 19 furthercomprising: administering allopurinol to the mammal.
 41. The method ofclaim 19 further comprising: hydrating the mammal.
 42. The method ofclaim 19 further comprising: administering urate oxidase to the mammal.43. The method of claim 19 further comprising: administering a steroidalagent to the mammal.
 44. The method of claim 43 wherein the steroidalagent is prednisone.
 45. The method of claim 19 further comprising:administering G -CSF to the mammal.
 46. The method of claim 30 furthercomprising: administering allopurinol to the mammal.
 47. The method ofclaim 30 further comprising: hydrating the mammal.
 48. The method ofclaim 30 further comprising: administering urate oxidase to the mammal.49. The method of claim 30 further comprising: administering a steroidalagent to the mammal.
 50. The method of claim 49 wherein the steroidalagent is prednisone.
 51. The method of claim 30 further comprising:administering G-CSF to the mammal.
 52. A kit for treating tumors,wherein components of the kit are in a divided or undivided container,said components comprising: spores of an anaerobic bacterium which istoxin-defective; an agent which collapses tumor vasculature.
 53. The kitof claim 52 wherein a toxin gene of a wild type form of the anaerobicbacterium is deleted in the spores of the anaerobic bacterium.
 54. Thekit of claim 52 further comprising a DNA damaging chemotherapeutic drug.55. The kit of claim 52 wherein the anaerobic bacterium is Clostridiumnovyi.
 56. The kit of claim 52 wherein the anaerobic bacterium isClostridium sordellii.
 57. The kit of claim 52 wherein the agent isvinblastine.
 58. The kit of claim 52 wherein the agent is vincristine.59. The kit of claim 52 wherein the agent is colchicine.
 60. The kit ofclaim 52 wherein the agent is combretastatin A-4.
 61. The kit of claim52 wherein the agent is dolastatin-10.
 62. The kit of claim 52 whereinthe agent is 5,6 dimethylxanthenone-4-acetic acid.
 63. The kit of claim54 wherein the DNA damaging chemotherapeutic drug is mitomycin C. 64.The kit of claim 54 wherein the DNA damaging chemotherapeutic drug iscytoxan.
 65. A kit for treating tumors, wherein components of the kitare in a divided or undivided container, said components comprising:spores of Clostridium novyi bacteria which are toxin-defective;dolastatin-10; and cytoxan.